WP3: Structural analysis of MAPS

Objectives: 
  1. Analyse the structure of membrane-anchored cytoskeleton assemblies designed in WP1-2 with high-resolution cryo-TEM to provide the feedback to the design (WP1-2).
  2. Study the dynamics of the membrane anchored assembly in real-time liquid-phase (LP) electron microscopy on the scaffold localization and formation of MAPS.
  3. Characterize the shape and dynamics of MAPS modified vesicles with cryo- electron tomography (3D cryo-TEM) and liquid phase TEM (LP-TEM), respectively.

To study the structure of the MAPS in their native hydrated state they will be anchored to supported lipid bilayers on various substrates used, e.g. for cryo-TEM, highly transparent graphene oxide (GOox) sheets while for LP-TEM they will be immobilized on Si3N4 liquid cell windows. Cryo-TEM will provide high resolution in the lateral directions (task 3.1), whole liquid phase TEM will be used to study the dynamics of the system.

After establishing the structure of the MAPS assemblies with cryo-TEM on graphene oxide supports, its formation will be studied using LP in-situ experiments. In LP-TEM experiments the bilayer membrane will be deposited on the flow cell where different components will be labelled either directly with gold nanoparticles (“undacagold” 0.8nm or “nanogold” 1.4 nm) or through gold-labelled streptavidin. LP-TEM and LP- STEM will be evaluated for imaging of different systems (task 3.2).

The effect of the protein reinforcement on the shape of the vesicles will be studied with 3D cryo-TEM while the dynamics of the liquid phase will be monitored with LP-TEM (task 3.3).